Immune checkpoint inhibitor (ICI) immunotherapies have revolutionized the treatment of solid cancers, but the success and efficacy of this line of therapy is often limited by treatment-induced immune toxicities, which have been reported to affect nearly every organ system with a wide spectrum of severity. These are termed immune- related adverse events (irAEs) and include complications such as myocarditis, hepatitis, colitis, pneumonitis, and rheumatoid arthritis. As ICIs become first- and second-line of cancer treatment in the years to come, irAEs are increasingly becoming the Achilles' heel of such treatments, and it is expected that the number of irAEs will continue rising and severely limit ICIs lifesaving potential unless we find solutions. While we can hypothesize that the irAE onset is a consequence of manipulating and targeting T-cell pathways through these ICIs and the breaking of tissue specific immune tolerance, the downstream mechanisms involved in driving and sustaining excessive immunotoxicity against normal organs remain poorly understood, resulting in limited treatment options. Notably, mice treated with ICIs do not develop such significant toxicities, which greatly limits their utility in understanding irAEs; this underscores the critical need for human translational research to elucidate underlying mechanisms, which is at the core of this proposal. This study outlines an integrative and unbiased experimental and analytical framework empowering the direct analyses of irAE patient's immune cells isolated from serially collected blood and matched inflamed tissue specimens to develop a detailed understanding of the molecular and cellular pathways involved in driving and sustaining the break in immune tolerance resulting in irAEs. To thoroughly analyze samples collected across a range of irAE complications and nominate shared and distinct intrinsic mechanisms between types of irAEs, we will use a combination of (i) cutting-edge single-cell RNA sequencing strategy to define the cellular and molecular perturbations characterizing irAEs, (ii) TCR and BCR repertoire analyses to establish the relevance of repertoire diversity as a clinical predictor, (iii) chromatin accessibility assay using ATAC-seq to better define the role of T-cell exhaustion and plasticity in irAEs, (iv) tissue imaging to map cellular ecosystem organization in inflamed tissue, and (v) profiling of secreted factors (e.g., cytokines, autoantibodies), circulating cell-free DNA methylation patterns, and immunophenotyping of cellular components in the blood that may track with the emergence and resolution of irAEs and thus could serve as biomarkers. Importantly, with these irAEs resembling idiopathic autoimmune diseases, this study also offers a rare window to study the exact moment when a patient's own immune system starts mounting an inflammatory response against normal tissue and may provide critical insights into human immune response regulation. Collectively, this proposal embodies a true bench-to-bedside evaluation of new disease entities that may identify culprit cellular components and molecules that could be targeted through `primary prevention' screening approach, or targeted after the onset of irAEs, without reducing the efficacy of the immunotherapy.